cis-Diamminedichloroplatinum(II) (cis-DDP) is a widely prescribed chemotherapeutic drug used to treat a variety of malignancies. A major limitation of cis-DDP treatment is recurrence and subsequent resistance. Resistance to cis-DDP treatment is, in part, a result of the ability to repair cis-DDP-DNA adducts. Understanding the basic mechanism of cis-DDP repair and the regulation of repair specific protein expression is essential to identify how cis-DDP resistance occurs and to develop effective therapies. Therefore, our goal is to determine the enzymatic pathway responsible for the repair of cis-DDP DNA adducts. Our approach is to purify proteins from mammalian cells, which are likely to be involved in repair of cis-DDP-DNA adducts based on their affinity for cis-DDP damaged DNA. These include human helicase E, damage recognition proteins (DRPs), a recently identified DNA dependent ATPase and damage incision proteins (DIPs). Extensive characterization of the individual proteins will be performed with a focus on how they interact with cis-DDP damaged DNA. DRPs and DIPs will also be characterized with respect to specificity for different types of DNA damage. Specifically, their binding and incision activities will be tested on thymine dimers, psoralen, 2-aminofluorine and cc1065 DNA adducts. Characterization of helicase E includes activity on cis-DDP and UV damaged DNA substrates and the ability to form a functional complex with DNA polymerase epsilon to perform repair synthesis. The effect of DRPs bound to damaged DNA on helicase activity will also be assessed. Initial characterization of ATPase-I will include determining if other DNA metabolizing activities, specifically helicase, ligase or nuclease, are associated with ATPase-I activity. In addition, the activity of ATPase-I will be assessed using a variety of synthetic DNA substrates designed to mimic structures found in DNA replication and repair. ATPase-I activity will also be measured using UV and cis-DDP damage DNA substrates. The effect of DRPs bound to damaged DNA on ATPase-I activity will also be evaluated to assess how the proteins interact. Antibodies will be raised against the two DRPs and ATPase-I and used to assess the role of each protein in an in vitro DNA repair assay and a Xenopus ooyte excision repair system. The level of expression of these proteins will be assessed in cis-DDP resistant and sensitive cell lines and XP cells. Immunoaffinity purification protocols will be established to aid in the purification of these proteins from cis-DDP resistant cell lines and XP cells, which will then allow direct comparison of the activities from the different cell lines.